Thermal sensation and pain use ion channels for detection of environmental cues. Thermal TRP channels, members of the transient receptor potential superfamily, are the principal detectors of thermal stimuli. These channels have a steep temperature dependence compared to other proteins. The long term goal of this research is to understand how the channels obtain their strong thermal sensitivity. We will focus on the vanilloid receptor TRPV1, a founding member of the thermal TRP subfamily. The channel plays a pivotal role in pain transduction and is abundantly expressed in peripheral sensory neurons where it appears to act as a gateway for detection and integration of noxious stimuli. Our previous research made progress in identifying the origin of thermal sensitivity of TRPV1, and showed that the channel contains modular thermal sensor domains in its N-terminus. We propose to take advantage of these findings to perform a comprehensive biophysical study on the fundamental mechanisms of temperature-dependent gating, using approaches that have proven successful for understanding other types of ion channel gating. Aim 1 focuses on the physical basis of thermal sensors. The hypothesis to be tested is that the N-terminal domain is responsible for distinct temperature phenotypes of TRPV1 homologs. By dissecting the molecular determinants of the phenotypic differences, we will identify the residues and subdomains contributing to temperature sensing. Aim 2 examines the interactions between sensing domains and subunits. We will test the cooperativity of thermal sensing between subunits, delineate the contribution of individual subunit thermal sensing events to channel opening, and probe the influence of thermal sensitivity by other stimuli. The results will unravel complex mechanisms by which TRPV1 achieves a dynamic thermal sensitivity for its physiological function over broad temperature ranges. Aim 3 addresses the coupling of the thermal sensor domain with the channel gate. We will test several regions throughout the channel and determine the allosteric mechanisms by which they control temperature activation. The results will illuminate the temperature-gating pathway in TRPV1 that links thermal sensing and gating. Our approach involves patch-clamp recording from recombinant channels in heterologous expression systems, combined with fast temperature stimulation and kinetic analysis to unravel the molecular events occurring during activation, along with mutagenesis to identify functional domains of the receptor. Thermal TRP channels are attractive ion-channel targets for the development of novel analgesic drugs that could act peripherally at nociceptors where pain is generated. With insight into how the channels function, the proposed studies will help prompt the selective drug development for treatment of pathologies such as thermal hyperalgesia due to inflammation, peripheral nerve injury, diabetes and herpes simplex.